Abstract
Prenatal hypoxia (PH) is a common feature of a suboptimal intrauterine environment affecting the development of fetuses. Whether PH leads to abnormal ovary development is not yet clear. This study investigated ovarian function in offspring exposed to PH and the potential underlying molecular mechanisms. SD female rats (n = 12 per group) at 9 weeks of age were housed in individual cages (21% O2). After the pregnant rats were exposed to hypoxia (10.5% oxygen) from embryonic day (E) 5 to E21, PH offspring were generated. All animals maintained normoxia during lactation. The number of follicles was counted in female offspring at 3 months under an optical microscope. The expression of Nobox, Gdf9, and Tets was detected by quantitative real-time polymerase chain reaction (PCR) and Western blot. Global DNA hydroxymethylation was measured by dot blot. The hydroxymethylation level of the Nobox gene was evaluated with an NGS-based multiple targeted CpG hydroxymethylation analysis method. Body weight and ovary weight were significantly decreased in the PH group compared with the control group. PH offspring have abnormal estrous cycle, decreased serum anti-Mullerian hormone (AMH), and increased serum follicle-stimulating hormone (FSH), and follicular atresia, which are consistent with the clinical manifestations in patients with ovarian dysfunction. In terms of mechanism, the expression of Nobox was significantly decreased in the PH group. Subsequent high-throughput sequencing results showed that the level of hydroxymethylation in the candidate region of the Nobox gene was reduced. Cultured cells treated with hypoxia exhibited lower levels of both 5hmC and Nobox, while vitamin C, a coactivator of Tets, rescued hypo-hydroxymethylation and increased the expression level of Nobox. This study indicated that PH could cause hypo-hydroxymethylation of Nobox through epigenetic regulation and may consequently contribute to ovarian dysfunction in adult rat offspring.
Highlights
The intrauterine environment plays an important role in shaping fetal development and impacting later health [1]
Bodyweight and ovary weight at 3 months were reduced in the Prenatal hypoxia (PH) group compared with the control group (P < 0.05, t test); there was no significant difference in the ratio of ovary weight to body weight between the two groups (Fig. 1a)
The serum anti-Mullerian hormone (AMH) level was significantly decreased (P < 0.01, t test), whereas the serum FSH level was significantly increased on the ovary morphology and percentage of follicles in rats. (CON, empty column, n = 10, PH, black column, n = 10). (D, diestrus; M, metestrus; E, estrus; P, proestrus; T, testosterone; AMH, anti-Müllerian hormone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; red arrow, corpus luteum, yellow arrow, primary follicle; green arrow, secondary follicle; black arrow, atretic follicle; blue arrow: granule cells). *P < 0.05, **P < 0.01, ***P < 0.001 PH vs. CON
Summary
The intrauterine environment plays an important role in shaping fetal development and impacting later health [1]. PH is associated with intrauterine growth restriction, fetal brain injury, and premature delivery [6–8]. PH has long-term adverse health effects on offspring’s health, namely, the nervous system, cardiovascular and metabolic disease, and so on [9–11]. Reproductive Sciences (2022) 29:1424–1436 dysfunction and metabolic disease, altered cardiac function, and increased susceptibility to ischemia–reperfusion injury [12–14]. These results are consistent with the fetal origins of adult disease (FOAD) theory proposed by David Barker [15]. The “Barker hypothesis” highlights the importance of intrauterine factors as contributors to FOAD, and recent investigations indicate that adverse intrauterine environments can cause changes in epigenetic modification and result in adult disease [16–18]
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